C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

In addition to m7 GpppN cap at their 5 -ends, virtually all eukaryotic mRNAs contain a poly(A) tail formed by template-independent addition of adenosine monophosphates residues to their 3'-terminal hydroxyl groups. The resulting 3 -poly(A) tail cooperates synergistically with the 5 -capping to stimulate translation with supra-additive potentiation, presumably by circularizing mRNA (62) . In addition, poly(A) tails promote stability of the transcripts, while deadenylation is a major rate-limiting step in mRNA decay (63, 64) . In contrast to the nuclear polyadenylation, the poly(A) tail of the C3P3-G1 is generated by the templatedependent transcription of a short 40 adenosine track from the DNA template, followed by a self-cleaving ribozyme sequence. The length of the poly(A) tail of the C3P3-G1 transcripts encoded by the templates is therefore much shorter than those of mammalian transcripts, which exit the nucleus with a uniform length of ∼250 nucleotides (65) (66) (67) (68) . As the length of the poly(A) tail is strongly correlated with the level of gene expression (69) , the polyadenylation from the C3P3-G1 expression system can be seen as sub-optimal. Further generations of the C3P3 system will improve the polyadenylation process, possibly by tethering a poly(A) polymerase to the C3P3 transcripts.